Functional analysis of the p300 acetyltransferase domain: the PHD finger of p300 but not of CBP is dispensable for enzymatic activity

Acetylation of nucleosomal histones is a major regulatory step during activ ation of eukaryotic gene expression. Among the known acetyltransferase (AT) families, the structure-function relationship of the GNAT superfamily is t he most well understood. In contrast, less information is available regardi ng mechanistic and regulatory aspects of p300/CBP AT function. In this pape r, we investigate in closer detail the structure and sequence requirements for p300/CBP enzymatic activity. Unexpectedly, we find that the PHD finger of p300, but not of CBP, is dispensable for AT activity. In order to identi fy residues involved in substrate or acetyl-coenzyme A (acetyl-CoA) recogni tion, we have introduced 19 different amino acid substitutions in segments that are highly conserved between animal and plant p300/CBP proteins. By pe rforming acetylation reactions with histones, a p53 peptide or the AT domai n itself, we define several residues required for histone and p53 substrate recruitment but not for acetyl-CoA binding. Finally, we show that identica l mutations in the p300 and CBP AT domain impair AT activity differently. T his latter result combined with the finding of a differential requirement f or the PHD finger provides evidence for structural differences between p300 and CBP that may in part underlie a previously reported functional special ization of the two proteins.